MORG1 (Mitogen-Activated Protein Kinase Organizer 1)
MORG1 and Its Role in the HIF Signaling
Besides its function as a scaffold protein of the MAPK pathway, MORG1 also plays an important role in the HIF (hypoxia-inducible factor) signaling (Fig. 2b). HIFs are oxygen-sensitive basic helix-loop-helix proteins, which consist of a labile α subunit (HIF-1α, HIF-2α, or HIF-3α) and a constitutively presented β (ARNT) subunit (Loeffler and Wolf 2015a). HIFs are the master transcription factors that enhance gene expression and regulate adaptive responses against tissue hypoxia (Loeffler and Wolf 2015a). Under hypoxic conditions, through reduced oxygen availability in the atmospheric air or in the context of many human diseases including cancer and kidney diseases, the α and β subunits heterodimerize to form a functional complex that translocates into the nucleus and transcriptionally activates myriad of genes involved in angiogenesis, glucose metabolism, cell proliferation, erythropoiesis, and apoptosis (Loeffler and Wolf 2015a). Under normoxia, specific prolyl hydroxylase domain proteins (PHDs) catalyze the oxygen-dependent hydroxylation of certain prolyl residues in HIF-α subunits resulting in polyubiquitylation and subsequent rapid proteasomal degradation (Loeffler and Wolf 2015a). Although each isoform of the three identified PHDs (PHD1, PHD2, and PHD3) displays its own tissue and cell line-specific expression pattern as well as its particular subcellular distribution, PHD3 (also named EGLN3) is distinct in many ways because it can mediate diverse cellular outcomes depending on the cell type and the extracellular cues (Hopfer et al. 2006). By screening a cDNA library with yeast two-hybrid assays, Hopfer et al. identified MORG1 as a PHD3-associated protein and confirmed the interaction in vitro and in vivo (Hopfer et al. 2006). They found that MORG1 displays a similar expression pattern as PHD3 and that both proteins perfectly co-localize within the cytoplasm and the nucleus (Hopfer et al. 2006). The characterization of the binding region of MORG1 offered that the binding of MORG1 to PHD3 occurs through a conserved region predicted to be on the top surface of one propeller blade (Hopfer et al. 2006). Whereas PHD3 has no impact on the function of MORG1 as an ERK activator in this system, HIF-mediated reporter gene activity is decreased by MORG1, and this effect is additive to almost basal levels by coexpression of PHD3 (Hopfer et al. 2006). MORG1 suppression, in turn, superinduced the HIF-mediated reporter gene activity in vitro as well as increased the basal HIF-1α and HIF-2α protein stability or rather decreased the HIF-α degradation in vivo, demonstrating that the scaffold protein MORG1 activates/stabilizes PHD3 and assists in the regulation of HIF-α protein (Fig. 2b) (Bondeva et al. 2013; Hopfer et al. 2006; Loeffler and Wolf 2015b).
Further Associations/Involvements of MORG1
In a comprehensive study of the human spliceosome, MORG1 was identified as one of the more than 200 associated proteins (Hegele et al. 2012). The spliceosome catalyze the pre-mRNA splicing and is a highly complex, dynamic, and protein-rich ribonucleoprotein complex that assembles de novo on each intron to be spliced (Hegele et al. 2012). It has been shown that MORG1, as a core protein, is abundant in the human spliceosomal C complex, in which the first of the two catalytic steps of splicing occurs (Hegele et al. 2012). A systematical analysis of human spliceosomal protein-protein interactions has shown that MORG1 interacts with SF3b49 as well as hPRP8, which is a U5 protein of large size (220 kDa) with multiple protein-protein interactions reflecting its central role as a scaffold protein within the spliceosome (Fig. 2c) (Hegele et al. 2012).
Another and very interesting involvement of MORG1 in an important cellular process has been recently reported. The study of Hayase et al. showed that MORG1 is involved in the formation of cell polarization in epithelial cells, which is crucial for morphogenesis (e.g., cyst formation) and function (e.g., tight junction development) (Hayase et al. 2013). The formation of apicobasal polarity in epithelial cells involves atypical protein kinase C (aPKC), which constitutively interacts with Par6, an evolutionarily conserved adaptor protein (Hayase et al. 2013). The Par6-aPKC complex is shown to translocate from the cytoplasm to the apical membrane, where the complex may be anchored with the apical membrane-integrated protein Crumbs3 (Crb3), which is reinforced with the apically localized small GTPase Cdc42 in the GTP-bound form (Hayase et al. 2013). For long time the mechanism for Par6-aPKC translocation to the apical membrane was unclear, but now it has been shown that the forced targeting of the complex to the apical surface is mediated by MORG1 (Fig. 2d) (Hayase et al. 2013). This conclusion is based on the findings that MORG1 directly binds not only to Par6 but also to Crb3, which facilitates Par6 binding to Crb3, leading to apical targeting of Par6-aPKC (Hayase et al. 2013). At the apical membrane Cdc42 increases the affinity of the Par6-aPKC complex to Crb3 and simultaneously promotes the dissociation of MORG1 from the Crb3-Par6-aPKC complex (Hayase et al. 2013). MORG1 released from the apical complex may redistribute to the cytoplasm to interact with another Par6-aPKC (Hayase et al. 2013). RNAi-mediated depletion of MORG1 results in mislocalization of Par6-aPKC to the cytoplasm and disruption of tight junction development in monolayer culture and cyst formation in 3D culture, emphasizing the crucial role of MORG1 in the formation of apicobasal polarity in epithelial cells (Hayase et al. 2013).
Role of MORG1 and Its Natural Antisense Transcript DHPS in Several Diseases
Several lines of evidence support the notion that MORG1, besides its physiological function, is also involved in the pathophysiology of various diseases. The involvement of MORG1 in kidney diseases has been described in animal models. Because of the embryonic lethality of homozygous MORG1 knockout mice, heterozygous MORG1 knockout mice were used to study the role of MORG1 in different models of acute and chronic kidney disease (Bondeva et al. 2013; Hammerschmidt et al. 2009; Loeffler and Wolf 2015b). Renal ischemia with reperfusion injury is a major cause of acute renal failure when pro-inflammatory and apoptotic processes in the kidney are activated and mice with reduced MORG1 expression are partially protected against kidney damage as a consequence of less inflammation and decreased apoptosis (Hammerschmidt et al. 2009). The same protective potential of reduced MORG1 expression was observed in another mouse model of acute kidney injury in which mice were exposed to systemic hypoxia (diminished oxygen in the environment) (Loeffler and Wolf 2015b). Whereas wild-type (MORG1+/+)mice showed a deterioration of renal function and significant increase in renal fibrosis, inflammation, and apoptosis as a result of diminished oxygen availability, kidneys of heterozygous MORG1 (MORG1+/−) knockout mice were protected against systemic hypoxia (Loeffler and Wolf 2015b). It is believed that the amelioration of acute renal injury in MORG1 heterozygous mice compared with MORG1 wild-type mice after renal ischemia with reperfusion as well as systemic hypoxia is caused by the stabilization of HIF-α protein when MORG1 activity is decreased (Hammerschmidt et al. 2009; Loeffler and Wolf 2015a, b). The a priori increased in basal levels of HIF-α, and its downstream target erythropoietin (EPO) in MORG1+/− mice is comparable with preconditioning or pharmacologic/genetic activation of HIF signaling, for which different studies the potential for the protection of kidneys against acute kidney injury have shown (Loeffler and Wolf 2015a). Although in chronic kidney disease, such as diabetic nephropathy, the tissue hypoxia is only one of the multiple pathological factors (i.e., angiotensin II, advanced glycation end products, and transforming growth factor β1), it has been demonstrated that angiotensin II differentially regulates MORG1 in kidney cells with and without involvement of MAPK (Bondeva et al. 2012), it is supposed that MORG1 is involved in the pathophysiology by acting predominantly as the scaffold protein of the HIF signaling rather than acting within the MAPK pathway. Moreover, recent published and unpublished data from our group show that heterozygosity of MORG1 ameliorates hallmarks of diabetic nephropathy, such as albuminuria, tubular damage, and tubulointerstitial fibrosis, primarily through stabilization and activation of HIF-2α (unpublished results and (Bondeva et al. 2013)).
MORG1 is not only expressed in kidney cells but also in the human brain in neurons, glial cells, and blood vessel walls (Haase et al. 2009). It has been discovered that MORG1 expression is complexly regulated in the ischemic human brain (reduced expression in neurons with ischemic damage but upregulation of the protein in astrocytes surrounding the penumbra (Haase et al. 2009). Interestingly, in a mouse model of focal cerebral ischemia-reperfusion, the stroke areas were smaller in heterozygous MORG1+/− mice compared with the MORG1+/+ wild-type (Stahr et al. 2012). It is believed that in MORG1 heterozygous mice a compensatory increase in MORG1 expression in astrocytes in the penumbra may negatively influence infarct volume and that these effects are independent of the PHD3-HIF1α axis (Stahr et al. 2012).
Whether MORG1 is also involved in craniosynostosis, a condition in which the cranial sutures prematurely fuse, remains unclear. Although a growing body of evidence in the literature supports the association of specific microdeletions in the small arm of chromosome 19 (19p13.12-19p13.2) and cranial suture dysmorphology, fully or partly deleted coding regions in the MORG1 gene affecting its expression are described only in some cases of craniosynostosis (Lyon et al. 2015). If MORG1 deletion is associated with the abnormal cranial vault development, then it is likely that MORG1 acts as a mediator of osteoclast dysregulation by disruption of the ERK signaling pathway, which is integral to osteoclast development and activity (Lyon et al. 2015).
The pathophysiological function of MORG1 as a module in the assembly of a multicomponent scaffold for components of the ERK-MAPK pathway has been additionally observed in acute myeloid leukemia (AML), gastric cancer, and other cancers (Su et al. 2012; Zhang et al. 2006). In AML the leukemogenic fusion protein AML1-ETO is one of the most frequent chromosomal abnormalities (Zhang et al. 2006). The chimeric protein AML1-ETO is generated by chromosomal translocation t(8;21) of the AML1 gene on chromosome 21 with ETO gene on chromosome 8 and induces both growth arrest and differentiation block on leukemic cells (Zhang et al. 2006). It has been shown that MORG1 is a target protein of AML1-ETO and its expression is downregulated in AML1-ETO-carrying leukemic cells (Zhang et al. 2006). Based on the knowledge that ERK-induced phosphorylation of AML1 is critical to AML1’s normal and physiological function as a crucial transcription factor for definitive hematopoiesis, it is postulated that the decrease of MORG1 might reduce the ERK activity and thus inhibit phosphorylational activation of AML1, which may become a way for the dominant-negative effect of AML1-ETO on wild-type AML1 function (Zhang et al. 2006).
MORG1 and its natural antisense transcript (NAT) deoxyhypusine synthase (DHPS) are transcribed from opposite strands of the same region on chromosome 19 in humans and form a “tail-to-tail” pairing pattern, including 113 nucleotides with full complementarity to their constitutive 3′UTRs (Su et al. 2012). NATs exist ubiquitously in mammalian genomes, are regulatory coding or noncoding RNAs, and play significant roles in physiological or pathological processes and regulate gene expression through direct interaction with the sense transcripts or indirect with other targets (Su et al. 2010). In gastric cancer mRNA and protein of MORG1 and DHPS are elevated concordantly, and they regulate one another in a bidirectional manner (Su et al. 2012). MORG1 and DHPS form an RNA duplex at overlapping 3′ untranslated regions, which mutually increased their stability and is required for the bidirectional regulation (Su et al. 2012). Furthermore, it has been shown that MORG1 and DHPS enhance the activation of ERK signaling in gastric cancer cells, which lead to the release and activation of the transcriptional factor E2F1 (Su et al. 2012). The aberrant activation of the ERK pathway is frequently observed in human cancers and knockdown, respectively; overexpression analysis with MORG1 and DHPS showed that both proteins drive gastric cancer pathophysiology by promoting cell proliferation (Su et al. 2012). In addition, the positive relationship between MORG1 and DHPS was also observed in other cancers and species (Su et al. 2012).
Finally, a very interesting finding regarding MORG1 and DHPS has been recently published: the finding is not related to disease but to the technological quality of pig meat (Zambonelli et al. 2013). Analysis of genomic regions with genes influencing pork pH, which is an important parameter for the quality assessment of fresh and seasoned meat products, offered SNPs in 3′UTR of the partly overlapping genes DHPS and MORG1 (Zambonelli et al. 2013). The oxygen reduction and the energy deficit of postmortem phase will lead to acidosis due to the anaerobic glycolysis increase that will cause a pH decrease, and the polymorphism detected in MORG1-DPHS overlapping region may activate this cascade with different efficiency between alleles to cause pH decline in the skeletal muscle cells during postmortem (Zambonelli et al. 2013). The identified association of the SNPs in MORG1-DHPS with meat pH could represent a new biomarker useful to improve pig meat quality (Zambonelli et al. 2013).
MORG1 has been identified as a scaffold protein, which is involved in several crucial physiological as well as pathophysiological processes. The importance of MORG1 is underlined by the embryonic lethality of constitutive MORG1 knockout mice, because of its role in controlling of a subset of ERK- and HIF-dependent biological responses. Interestingly, MORG1 is also responsible for the apical differentiation in epithelial cells and is part of the human spliceosome. On the other hand, increasing evidence suggests that MORG1 plays also an important role in many human diseases. Thus, MORG1 might serve as a novel therapeutic strategy for the treatment of, e.g., kidney diseases and cancer.
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